Genetation of beta-amyloid (Abeta) from native precursor protein (APP) and its accumulation which appear as senile plaque and amyloid angiopathy in human brain are essentially associated with normal aging and pathophysiology of Alzheimer's disease. Processing of APP and formation of Abeta-bearing peptides were comparatively analyzed between normal lymphoblastoid cells and those derived from familial AD patients. 68 kDa serine protease was purified from culture medium of AD cells, which cleaves oligopeptides at the Abeta-N-terminus. Though this protease cleaves native LAPP in the vicinity of Abeta-N- terminus, native brain APP was not degraded, suggesting a strict tissue specificity in APP proteolysis. I searched for activities capable of cleaving native brain APP in human hippocampus, and HBP49 proteolytic activity was isolated and characterized, and the molecular cloning clarified as being a novel protease. Biochemical and molecular biological analyses suggested a physiological function of the protease in brain APP degradation and generation of Abeta peptides, and a possible role of sulfated glycocon-jugates attached to brain APP in the proteolysis. Immunohistochemical study showed its expression in neuronal perikarya, and some unique lesions with the immunoreactivity were found in brains of sporadic AD patients.平成9年度研究実績から、ヒト天然APPに対する切断活性特に中性pH域におけるβアミロイド形成(切り出し)活性を脳海馬より探索した(論文1)。リンパ球より精製した68Kセリンプロテアーゼはリンパ球APP(LAPP)を切断し14kDaのβアミロイド含有ペプチドを形成するが、予想に反し海馬由来APPを切断しなかった(論文2)。ヒト脳由来APPはAPP695のほかLAPPをコア蛋白としコンドロイチン硫酸をもつプロテオグリカンの一種アピカンからなり、LAPPからなるリンパ球と分子構造を異にする。セクレターゼ群に著明な組織特異性があると推定されることより、ヒト脳(海馬)からの同活性の探索・精製を行う必要性が生じた。リガンド特異的アフィニティークロマトグラフィーと非変成ゲル電気泳動法を用い、各脳画分の天然APP分解活性を抗Aβ抗体をもちいたWestern法によりアッセイした。その結果、脳APPをAβ-N端近傍および他数箇所を切断する活性(HBP40プロテアーゼ)を見いだし、単離精製した。また本プロテアーゼに対する特異抗体を用い遺伝子単離、cDNA解析、免疫組織化学的解析(アルツハイマー病脳を含む)を行った(投稿中)。